DESCRIPTION. Resistance to antibiotics is widespread among pathogenic bacteria and exacts a high cost on society. It is critically important that we develop strategies to prolong the clinical usefulness of the existing antibiotics. It is our view that the key to overcoming resistance to antibacterials is a full knowledge of the mechanisms of such resistance. This detailed knowledge should ultimately be instrumental in designing new antibiotics or redesigning old ones that circumvent the resistance problem in bacteria. A focus of this grant application is on the mechanism of resistance to beta-lactam antibiotics in Staphylococcus aureus, an important pathogen that has developed elaborate mechanisms for resistance by expressing both beta- lactamases, enzymes that hydrolytically destroy these antibiotics, or a penicillin-binding protein (PBP 2a) that is not readily inhibited by beta-lactam antibiotics. Three specific Aims are proposed. Specific Aim 1 is to study the process of interactions of beta-lactam antibiotics with the integral membrane protein BlaR1 of S. aureus. The beta-lactam sensor domain of this protein detects the presence of the antibiotic, a signal is transduced to the cytoplasm, where a protease domain of the protein is activated. The activated protease degrades (directly or indirectly) the represser for genes that manifest in expression of antibiotic resistance enzymes, the beta-lactamase and PBP 2a. The study for the full details of these processes is outlined. Specific Aim 2 proposes to investigate the properties of the beta-lactam sensor domain of a related protein, MecR1. The studies will delineate the mechanistic differences that are seen clinically in the onset of antibiotic resistance via this protein compared to the corresponding domain of BlaR1. Specific Aim 3 will follow up on the discovery in the Mobashery lab of a set of molecules that inhibit all three classes of serine- dependent beta-lactamases and also the BlaR1 protein. This type of enzyme inhibitor is expected to reverse the resistance phenotype in bacteria, rendering the the organisms susceptible to known beta-lactam antibiotics. RELEVANCE. Antibiotic resistance is a serious clinical problem with important societal cnsequences. The proposed work is to study how resistance to beta-lactam antibiotics has developed. The understanding of the mechanistic details should guide the way in devising strategies to circumvent the problem.